A Catalog of over 5,000 Metagenome-Assembled Microbial Genomes from the Caprinae Gut Microbiota

ABSTRACT Most microbiome studies regarding the ruminant digestive tract have focused on the rumen microbiota, whereas only a few studies were performed on investigating the gut microbiota of ruminants, which limits our understanding of this important component. Herein, the gut microbiota of 30 Caprinae animals (sheep and goats) from six provinces in China was characterized using ultradeep (>100 Gbp per sample) metagenome shotgun sequencing. An inventory of Caprinae gut microbial species containing 5,046 metagenomic assembly genomes (MAGs) was constructed. Particularly, 2,530 of the genomes belonged to uncultured candidate species. These genomes largely expanded the genomic repository of the current microbes in the Caprinae gut. Several enzymes and biosynthetic gene clusters encoded by these Caprinae gut species were identified. In summary, our study extends the gut microbiota characteristics of Caprinae and provides a basis for future studies on animal production and animal health. IMPORTANCE We constructed a microbiota catalog containing 5,046 MAGs from Caprinae gut from six regions of China. Most of the MAGs do not overlap known databases and appear to be potentially new species. We also characterized the functional spectrum of these MAGs and analyzed the differences between different regions. Our study enriches the understanding of taxonomic, functional, and metabolic diversity of Caprinae gut microbiota. We are confident that the manuscript will be of utmost interest to a wide range of readers and be widely applied in future research.

author can combine and use these data, although the author generated a deep sequence data for each sample. Then we can get a relatively comprehensive view of ruminant fecal microbiome. L29-33. The author got MAGs, so what is the findings of these MAGs? But not generally describe the identification of MAGs. The enzymes and biosynthetic gene clusters are common results for metagenome study. What are the new findings and innovation? L33. How the above results support co-evolution and feed efficiency? Make no sense. L41-42. "Gut microbiota is considered to play a pivotal role in the energy conversion process of ruminants.". Rumen microbiome is much more important for energy harvest, although the lower gut microbiome is also critical for host healthy. L43. How the GIT microbiome can enhance nutrient intake? List the evidence. L47-54. Previous studies have reported the microbial gene catalog and constructed MAGs, so what is the significance of this study? It is accepted that rumen microbiome is complex than gut (usually represented by fecal samples) microbiome of monogastric animal. So how to conclude a high complexity of the gut microbiota of ruminants? What is unknown information based on the previous reports? Please give more supports for this hypothesis. L58. References for "Caprinae animals have species polymorphism, including goats, sheep, antelopes, etc.". L70-72. The environmental factors affected rumen and fecal microbiome. Why the author uses these ruminant species or breeds from only six provinces? Does these samples have clear representativeness, or can support the hypothesis in L58-69? L86. The threshold of sequencing coverage and GC content and identical taxonomic assignment. L97-98. Which version of GTDB? This affected the novelty of results. L114-115. The author identified novel species using NCBI database. However, the method section indicated that "21,315 highquality MAGs were downloaded from four publicly available animal gut microbiota datasets [9,11,33,34], which represented the most comprehensive animal gut microbiota genome data available to date.". Why? Is there a representativeness for animal gut microbiome? The human and other mammals (pig) have generated a much greater database of MAGs. L121-123. Why the genomes change? L123-164. The author aimed to extend the phylogenetic tree of microorganisms using 4 databases based on the 3,306 SGBs. First, the author should include more MAGs database from other mammals, human and non-human primate. Second, the these SGBs were obtained from bins, but not culturable microorganisms, and its taxonomic classification is unclear. Thus, there maybe common MAGs form different database, and the author should combine these database together, and then analyzed the distribution. Third, this study generated MAGs from fecal samples, however, the database RUG were from rumen, and RGIG were from whole GIT of 7 ruminant species. The microbial profile must differ. The comparison makes no sense. Forth, please provide the value for the proportions. Fifth, be careful to use habitat and ecosystem. L137. ruminant cattle? L166-170. The author constructed the phylogenetic tree of Akkermansia. Based on the results in figure 2c, it seems that some MAGs showed very close phylogenetic relationship on nodes. So what is the node support? Moreover, a recent study has analyzed 112 Akkermansia muciniphila strains based on phylogenetic tree (10.1128/spectrum. . This indicate the database used in this study is in completed. Please defined "crossly presented". L193-196. It is well known that the taxonomic classification absolutely affected the functional profile. The results for many studies have demonstrated the function of different phyla or families were distinct. So it is unreliable to get the conclusion "These differences in MAGs led to huge differences in the functional potential of the microbiota in the Caprinae gut". Comparing the results with fecal microbiome of other ruminants is warranted. L204. Please define KEGG level B or C. L208. Define a lot of. L210. encoded Valine? Define a large numbers. L259. gut MAGs. L184-279. This study explored the functional profiles by describing and quantifying KEGG, CAZymes, BGCs and antibioticresistant genes. This is important, but did not give a deeper insight to these MAGs, particular to the host-specific and novel MAGs. A comparative genome analysis will be helpful. L282. A significantly geographical effect was observed on bacterial community. So the bacterial community were based on MAGs or taxonomic classification? L287-296. The results showed species, altitude, and sex were responsible for 16.1%, 8.8% and 8.7% of variations, respectively. Although a species must have a relative abundance of 0.01% in at least 50% of samples from a region to be considered universal in samples from that region. However, only 5 Tibet samples from 4 ruminant species, this will affect the comparison and may mask the real effect of hosts. L294. Change region to geographical region. L296. Change 260 to Two hundred and sixty. L302-303. Again, all Tibet samples (n = 5) were compared with non-Tibet samples (n = 25). This comparison is difficult to reflect the real difference. L317-318. Can bacteria synthesize bile acids? L327-333. Repeat results, condense. L334-336. The author only compared the different database. Because the size of database is different, thus the largely complemented and extended the existing reference can not reach. Combining these dataset together, and then analyze the taxonomic classification of raw reads, this can demonstrate the supplement to previous reference. L337-338. The number of animals is more important than sequencing depth. L342-344. Remove. L345-346. A higher abundance of Ruminococcaceae in this study in comparison of gut core microbiota in other animals. How to define core microbiota? And thus can conclude the complex of caprinae gut. L348. What is other animals? L349-350. Although the sheep are thought to be typically herbivores, their food sources in the wild may actually be more complex. Reference. L357. What is "these bacteria"? L358-359. "which may explain the source of some members and how the gut microbiome is shaped." What is some members? L361. What is "The unique taxonomic characteristics"? L363. "and this process occurs later than other gut bacteria". Make no sense. L369-370. The species-level diversity of these bacteria may suggest that they have evolved longer in the animal gut than in humans. Make no sense. L376-383. Meaningless. Discuss the novel findings, such as the changed genome size of MAGs from different phyla. L386-388. The diversity indices were not significantly changed, so how can conclude "This suggests that the geographic heterogeneity of gut microbiota may be caused by some species that metabolize specific substrates."? L392-397. Provide evidences. L449. Which kit? The DNA extraction methods have a clear effect on sequencing quality. L464. Is there a Procapra picticaudata genome in NCBI database?
Reviewer #2 (Comments for the Author): Comments: In this manuscript, the authors constructed metagenome-assembled reference genome catalog from Caprinae gut microbiota using ultra-deep metagenomics sequencing. This topic is extreme important nowadays because it is sooner and easier to acquire high quality genome of uncultured bacteria than traditional isolation and sequencing pipeline. Overall, the paper is well written (although the English could be improved, it was not a significant impediment to understanding), the experiments well designed, and the statistical analysis relevant and robust. I have only minor suggestions for improvement/clarification. 1. Line 94-95: the author clustered the MAG to establish SGB using 95% ANI. However, in material and methods, Line 481-482, 99% ANI was used in dRep to perform dereplication which was normally used to establish strain level cluster of MAG. Please clarify this. 2. Line 468-469: single sample assembly strategy was employed in this manuscript to acquire contig catalog. For metagenomics sequencing, co-assembly is also widely used to further refine and improve the assembly output of single sample assembly strategy, especially for ultra-deep metagenome sequencing. And this may have considerable effect on downstream binning output. Please clarify this issue.

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In this study, Zhang et al. examined the Caprinae fecal microbiome by constructing MAGs. However, the results are limited by sample size from 4 species. Moreover, the data analysis and writing need a greater improvement. L4-6. Make a consistent style of author name. L26. Gut is a very general definition, including ileum, cecum, colon and so on. Which region is focused on this study? and why? L27. Based on the information in methods: fecal samples from four Caprinae species (Capra hircus, n = 18; Ovis aries, n = 9; Pantholops hodgsonii, n = 2; Procapra picticaudata, n = 1 from six provinces of China (Anhui, Jilin, Guangxi, Shandong, Shanxi, and Tibet)) were collected (Table S1).", the author only analyzed the fecal microbiome using shotgun sequencing. How to represent gut? Ruminant includes many species. Why this study only use goat and sheep animals? Only 1 sample were obtained from Procapra picticaudata. Recently, fecal metagenomes were also reported by many studies. It is appreciated if the author can combine and use these data, although the author generated a deep sequence data for each sample. Then we can get a relatively comprehensive view of ruminant fecal microbiome. L29-33. The author got MAGs, so what is the findings of these MAGs? But not generally describe the identification of MAGs. The enzymes and biosynthetic gene clusters are common results for metagenome study. What are the new findings and innovation? L33. How the above results support co-evolution and feed efficiency? Make no sense. L41-42. "Gut microbiota is considered to play a pivotal role in the energy conversion process of ruminants.". Rumen microbiome is much more important for energy harvest, although the lower gut microbiome is also critical for host healthy. L43. How the GIT microbiome can enhance nutrient intake? List the evidence. L47-54. Previous studies have reported the microbial gene catalog and constructed MAGs, so what is the significance of this study? It is accepted that rumen microbiome is complex than gut (usually represented by fecal samples) microbiome of monogastric animal. So how to conclude a high complexity of the gut microbiota of ruminants? What is unknown information based on the previous reports? Please give more supports for this hypothesis. L58. References for "Caprinae animals have species polymorphism, including goats, sheep, antelopes, etc.". L70-72. The environmental factors affected rumen and fecal microbiome. Why the author uses these ruminant species or breeds from only six provinces? Does these samples have clear representativeness, or can support the hypothesis in L58-69? L86. The threshold of sequencing coverage and GC content and identical taxonomic assignment. L97-98. Which version of GTDB? This affected the novelty of results. L114-115. The author identified novel species using NCBI database. However, the method section indicated that "21,315 high-quality MAGs were downloaded from four publicly available animal gut microbiota datasets [9,11,33,34], which represented the most comprehensive animal gut microbiota genome data available to date.". Why? Is there a representativeness for animal gut microbiome? The human and other mammals (pig) have generated a much greater database of MAGs. L121-123. Why the genomes change? L123-164. The author aimed to extend the phylogenetic tree of microorganisms using 4 databases based on the 3,306 SGBs. First, the author should include more MAGs database from other mammals, human and non-human primate. Second, the these SGBs were obtained from bins, but not culturable microorganisms, and its taxonomic classification is unclear. Thus, there maybe common MAGs form different database, and the author should combine these database together, and then analyzed the distribution. Third, this study generated MAGs from fecal samples, however, the database RUG were from rumen, and RGIG were from whole GIT of 7 ruminant species. The microbial profile must differ. The comparison makes no sense. Forth, please provide the value for the proportions. Fifth, be careful to use habitat and ecosystem. L137. ruminant cattle? L166-170. The author constructed the phylogenetic tree of Akkermansia. Based on the results in figure 2c, it seems that some MAGs showed very close phylogenetic relationship on nodes. So what is the node support? Moreover, a recent study has analyzed 112 Akkermansia muciniphila strains based on phylogenetic tree (10.1128/spectrum.02439-21). This indicate the database used in this study is in completed. Please defined "crossly presented". L193-196. It is well known that the taxonomic classification absolutely affected the functional profile. The results for many studies have demonstrated the function of different phyla or families were distinct. So it is unreliable to get the conclusion "These differences in MAGs led to huge differences in the functional potential of the microbiota in the Caprinae gut". Comparing the results with fecal microbiome of other ruminants is warranted. L204. Please define KEGG level B or C. L208. Define a lot of. L210. encoded Valine? Define a large numbers. L259. gut MAGs. L184-279. This study explored the functional profiles by describing and quantifying KEGG, CAZymes, BGCs and antibiotic-resistant genes. This is important, but did not give a deeper insight to these MAGs, particular to the host-specific and novel MAGs. A comparative genome analysis will be helpful. L282. A significantly geographical effect was observed on bacterial community. So the bacterial community were based on MAGs or taxonomic classification? L287-296. The results showed species, altitude, and sex were responsible for 16.1%, 8.8% and 8.7% of variations, respectively. Although a species must have a relative abundance of 0.01% in at least 50% of samples from a region to be considered universal in samples from that region. However, only 5 Tibet samples from 4 ruminant species, this will affect the comparison and may mask the real effect of hosts. L294. Change region to geographical region. L296. Change 260 to Two hundred and sixty. L302-303. Again, all Tibet samples (n = 5) were compared with non-Tibet samples (n = 25). This comparison is difficult to reflect the real difference. L317-318. Can bacteria synthesize bile acids? L327-333. Repeat results, condense. L334-336. The author only compared the different database. Because the size of database is different, thus the largely complemented and extended the existing reference can not reach. Combining these dataset together, and then analyze the taxonomic classification of raw reads, this can demonstrate the supplement to previous reference. L337-338. The number of animals is more important than sequencing depth. L342-344. Remove. L345-346. A higher abundance of Ruminococcaceae in this study in comparison of gut core microbiota in other animals. How to define core microbiota? And thus can conclude the complex of caprinae gut. L348. What is other animals? L349-350. Although the sheep are thought to be typically herbivores, their food sources in the wild may actually be more complex. Reference. L357. What is "these bacteria"? L358-359. "which may explain the source of some members and how the gut microbiome is shaped." What is some members? L361. What is "The unique taxonomic characteristics"? L363. "and this process occurs later than other gut bacteria". Make no sense. L369-370. The species-level diversity of these bacteria may suggest that they have evolved longer in the animal gut than in humans. Make no sense. L376-383. Meaningless. Discuss the novel findings, such as the changed genome size of MAGs from different phyla. L386-388. The diversity indices were not significantly changed, so how can conclude "This suggests that the geographic heterogeneity of gut microbiota may be caused by some species that metabolize specific substrates."? L392-397. Provide evidences. L449. Which kit? The DNA extraction methods have a clear effect on sequencing quality. L464. Is there a Procapra picticaudata genome in NCBI database?